1. Fitting EM maps into X-ray Data Alexei Vagin York Structural Biology Laboratory University of York

2. EM  X-ray ( P2 ) --> MR --> H2OH2O H2OH2O

3. Molecular Replacement Cell Model 1. Rotation function 2.Translation function

4. Can we find solution? Weight of low resolution grows Low high Model Similarity 0.1 0.2 0.3 0.4 0.5 Weight of high resolution grows Low high Model size 0.5 0.4 0.3 0.2 0.1 Very likely Very unlikely EM model

5. Information in X-ray and EM must overlap s Resmin X-ray Resmax EM

6. Example 1 Groel-ATP7-Groes Map: EMD-1180 Fitted atomic model: PDB 2c7c X-ray: PDB 1sx4 ( 40 - 3 A )

7. EM map as model

8. EM map (sfcheck) from grid

9. EM map (sfcheck) 7A

10. Information in X-ray and EM must overlap s Resmin X-ray (40A) Resmax EM (7A)

11. ∆Ph =⃒Ph xray - Ph em ⃒ MR solution (EM model) ∆Ph =⃒Ph xray - Ph model ⃒ MR solution (fitted atomic model) 50 o 90 o Res: 11.1 9.1 7.7 6.7 5.9 5.3 4.8 4.3 4.0 A ∆Ph

12. Map ( F obs Ph em ) 6Å

13. What we can do with these phases 1. Help to find HA positions in the derivative 2. Extend phases by averaging electron density 3. Try to fit some fragments ( helix )

14. EM map (4A) Modified map Averaging and Solvent flattening

15. Map (F obs Ph em ) 4Å Modified map

16. ∆Ph =⃒Ph xray - Ph em ⃒ MR solution (EM model) ∆Ph =⃒Ph xray - Ph model ⃒ MR solution (fitted atomic model) ∆Ph =⃒Ph xray - Ph aver ⃒ MR solution (after DM) 50 o 90 o Res: 11.1 9.1 7.7 6.7 5.9 5.3 4.8 4.3 4.0 A ∆Ph

17. Free atom model

18. ∆Ph =⃒Ph xray - Ph em ⃒ MR solution (EM model) ∆Ph =⃒Ph xray - Ph model ⃒ MR solution (fitted atomic model) ∆Ph =⃒Ph xray - Ph model_ref ⃒ MR solution (refined atomic model) ∆Ph =⃒Ph xray - Ph free ⃒ MR solution (free atom model after DM) 50 o 90 o Res: 9.4 7.4 6.1 5.2 4.5 4.0 3.6 3.3 3.0 A ∆Ph

19. Map (F obs Ph em ) 3Å Map (F obs Ph free ) 3Å (Free atom model after DM)

20. Map (F obs Ph free ) 3Å (Free atom model after DM)

22. Example 2 X-ray structure of GP6 protein of phage SPP1 ( 3.4 A ) Fred Antson at al. University of York

23. EM map as model

24. EM map (sfcheck 1) from grid

25. EM map (sfcheck 2) 8A

26. Information in X-ray and EM must overlap s Resmin X-ray (40A) Resmax EM (8A)

27. ∆Ph =⃒Ph xray - Ph em ⃒ MR solution (EM model) 50 o 90 o Res: 10.3 8.3 6.9 6.0 5.2 4.7 4.2 3.8 3.5 A ∆Ph

28. Map ( F obs Ph em ) 7Å

29. What we can do with these phases 1. Help to find HA positions in the derivative 2. Extend phases by averaging electron density 3. Try to fit some fragments ( helix )

30. ∆ Ph =⃒Ph xray - Ph em ⃒ MR solution (EM model) ∆ Ph =⃒Ph xray - Ph aver ⃒ MR solution (after DM) 50 o 90 o Res: 10.3 8.3 6.9 6.0 5.2 4.7 4.2 3.8 3.5 A ∆Ph

31. Free atom model model was used only to compute initial phases

32. ∆ Ph =⃒Ph xray - Ph em ⃒ MR solution (EM model) ∆ Ph =⃒Ph xray - Ph aver ⃒ MR solution (after DM) ∆ Ph =⃒Ph xray - Ph free ⃒ MR solution (free atom model after DM) 50 o 90 o Res: 10.3 8.3 6.9 6.0 5.2 4.7 4.2 3.8 3.5 A ∆Ph

33. Final map 3.5 A

35. Map ( F obs Ph em ) Final map

36. What we can do with these phases 1. Help to find HA positions in the derivative 2. Extend phases by averaging electron density 3. Try to fit some fragments ( helix )

37. Fitting 17 x 13 helixes into final map by SAPTF

38. Final map 3.5 A and helixes

39. X-ray model and helixes

40. Model: helix_17x13.pdb 1.Refinement 2.Compute map for refined model 3.Combine final map and map from model Final map Combine map

41. ∆ Ph =⃒Ph xray - Ph em ⃒ MR solution (EM model) ∆ Ph =⃒Ph xray - Ph aver ⃒ MR solution (after DM) ∆ Ph =⃒Ph xray - Ph free ⃒ MR solution (free atom model after DM) ∆ Ph =⃒Ph xray - Ph comb ⃒ combine map 50 o 90 o Res: 10.3 8.3 6.9 6.0 5.2 4.7 4.2 3.8 3.5 A ∆Ph

42. The end http://www.ysbl.york.ac.uk/~alexei/

43. SOLUTION_CHECK: Are two solutions identical? Alexei Vagin York Structural Biology Laboratory University of York

44. It is trivial. X test [T origin ] [T symm_op ] Y test X target Alignment, Fitting Orientation and position differences

45. The end